The beta cells of the pancreas produce and secrete insulin in response to increasing blood glucose levels and this process is essential for maintaining normal glucose homeostasis. Defects in insulin production and secretion result in type 1 as well as type 2 diabetes. Although, three beta-cell specific transcription factors (Pdx-1, MafA, and NeuroD1) have been implicated in glucose regulation of insulin production, the exact molecular mechanisms by which glucose modulates the function of these transcription factors remain unknown. The long-term goal of this research is to gain a detailed understanding of the mechanisms and pathways leading to glucose induction of insulin gene expression via modulation of the transcription factors Pdx-1, MafA, and NeuroD1. Our previous findings suggest that glucose regulates the function of the these transcription factors by different mechanisms and pathways to ensure a tight control of insulin gene expression in pancreatic beta cells. Based on our preliminary data, we hypothesize that glucose regulates insulin gene expression by 1) influencing the interaction of Pdx-1 with other transcriptional regulators;2) controlling MafA expression via O-linked GlcNAc modification of proteins;and 3) regulating the nucleo-cytoplasmic shuttling of NeuroD1. We propose to test this hypothesis in cultured pancreatic beta cell lines as well as isolated pancreatic islets using various biochemical, molecular, and cell biological, and immunological techniques to identify the critical pathways and players involved in glucose regulation of beta-cell specific transcription factors. The information obtained from our studies will be instrumental in the design of new therapies to treat and prevent diabetes, associated with defects in insulin production.